Eukaryotic proteins are synthesized within the endoplasmic reticulum (ER), are delivered from the ER to the Golgi complex for post-translational processing and sorting, and are transported from the Golgi to specific intracellular and extracellular destinations. This intracellular and extracellular movement of protein molecules is termed vesicle trafficking. Trafficking is accomplished by the packaging of protein molecules into specialized vesicles which bud from the donor organelle membrane and fuse to the target membrane.
Specialized cell types utilize specific vesicle trafficking routes. For instance, in endocrine glands, hormones and other secreted proteins are delivered to secretory granules for exocytosis through the plasma membrane to the cell exterior. In macrophages, peroxidases and proteases are delivered to lysosomes. In fat and muscle cells, glucose transporters are stored in vesicles which fuse with the plasma membrane in response to insulin stimulation.
Numerous proteins are necessary for the formation, targeting, and fusion of transport vesicles and for the proper sorting of proteins into these vesicles. The vesicle trafficking machinery includes coat proteins which promote the budding of vesicles from donor membranes; vesicle- and target-specific identifiers (v-SNAREs and t-SNAREs) which bind to each other and dock the vesicle to the target membrane; and proteins which bind to SNARE complexes and initiate fusion of the vesicle to the target membrane (SNAPs).
Vesicles in the process of budding from the ER and the Golgi are covered with a protein coat similar to the clathrin coat of endocytotic vesicles. The protein coat is assembled from cytosolic precursor molecules and is confined to budding regions of the organelle membrane. The coat protein (COP)-coated vesicles are uncoated after budding is complete to allow fusion of the vesicle to the donor membrane.
The "pinching off" of the nascent vesicle bud requires a process distinct from coat assembly. Periplasmic fusion, which is membrane fusion initiated from the cytoplasmic side of the bud, may be mediated by integral membrane proteins present in the transport vesicles (Rothman, J. E. (1994) Nature 372:55-63). A membrane protein isolated from COP-coated vesicles of chinese hamster ovary (CHO) cells was found to belong to a family of homologous 24 kdal proteins, known as the p24 family, from ER and Golgi membranes of a broad range of organisms (Stamnes, M. A. et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92:8011-8015).
The p24 family consists of integral membrane proteins which contain a single transmembrane domain located near the C-terminus. All p24 proteins possess a phenylalanine residue located in the cytoplasmic C-terminal portion of the molecule near the transmembrane segment. In all known mammalian p24 proteins, the conserved phenylalanine is followed by two or three basic residues near the C-terminus (Fiedler, K. et al. (1996) Science 273: 1396-1399). p24 proteins bind to various subunits of the COP-coatmer complex, depending on the arrangement of the C-terminal basic residues (Fiedler et al., supra).
A yeast p24 homolog yp24A (also known as Emp24p) isolated from ER-derived COP-coated vesicles is required for the efficient transport of a subset of secretory proteins from the ER to the Golgi (Stamnes et al., supra; Schimmoller, F. et al. (1995) EMBO J. 14:1329-1339). Electron microscopy of yeast cells lacking functional p24A reveals a decrease in steady state vesicle accumulation, which indicates that yp24A is necessary for efficient vesicle budding (Stamnes et al, supra).
Since transport of only a subset of yeast secretory proteins is affected in yp24A mutant cells, Schimmoller, et al. (supra) propose that different yeast p24 homologs may recognize and capture distinct, possibly overlapping sets of proteins into secretory vesicles.
Similarly, Stamnes et al. (supra) and Rothman et al. (1996; Science 272:227-234) speculate that p24 homologs may serve as "cargo receptors", selecting proteins for inclusion in budding COP-coated vesicles.
Other members of the evolutionarily related p24 protein family have been cloned from rat and human (Blum, R. et al. (1996) J. Biol. Chem. 271:17183-17189). Rat p24A is abundantly expressed in pancreas, consistent with the proposed role of p24 in the sorting and directing of proteins within the secretory pathway. Furthermore, a protein identified in a human glioblastoma cell line shows significant homology to the p24 family (Gayle, M. A. et al. (1996) J. Biol. Chem. 271:5784-5789). The protein was identified based on its ability to bind to the type I interleukin-1 (IL-1) receptor homolog T1/ST2, yet shows no biological activity in IL-1 or T1/ST2 receptor-based assays (Gayle, supra). Therefore, the putative T1/ST2 binding protein may be another member of the human p24 family.
The etiology of numerous human diseases and disorders can be attributed to defects in the trafficking of proteins to organelles or the cell surface. Defects in the trafficking of membrane-bound receptors and ion channels are associated with cystic fibrosis (cystic fibrosis transmembrane conductance regulator; CFTR), glucose-galactose malabsorption syndrome (Na.sup.+ /glucose cotransporter), hypercholesterolemia (low-density lipoprotein (LDL) receptor), and forms of diabetes mellitus (insulin receptor). Abnormal hormonal secretion is linked to disorders including diabetes insipidus (vasopressin), hyper- and hypoglycemia (insulin, glucagon), Grave's disease and goiter (thyroid hormone), and Cushing's and Addison's diseases (adrenocorticotropic hormone; ACTH).
Cancer cells secrete excessive amounts of hormones or other biologically active peptides. Disorders related to excessive secretion of biologically active peptides by tumor cells include: fasting hypoglycemia due to increased insulin secretion from insulinoma-islet cell tumors; hypertension due to increased epinephrine and norepinephrine secreted from pheochromocytomas of the adrenal medulla and sympathetic paraganglia; and carcinoid syndrome, which includes abdominal cramps, diarrhea, and valvular heart disease, caused by excessive amounts of vasoactive substances (serotonin, bradykinin, histamine, prostaglandins, and polypeptide hormones) secreted from intestinal tumors. Ectopic synthesis and secretion of biologically active peptides (peptides not expected from a tumor) includes ACTH and vasopressin in lung and pancreatic cancers; parathyroid hormone in lung and bladder cancers; calcitonin in lung and breast cancers; and thyroid-stimulating hormone in medullary thyroid carcinoma.
Polynucleotides encoding novel human p24 vesicle trafficking proteins and the molecules themselves provide a means to investigate vesicle trafficking and secretion under normal and disease conditions. Discovery of novel p24 vesicle trafficking proteins satisfies a need in the art by providing new compositions useful in diagnosing and treating disorders associated with abnormal vesicle trafficking.